DESCRIPTION: All eukaryotic cells depend on extracellular nutrients and environmental cues for long-term survival. This constant flow of information frequently leads to compensatory remodeling of the plasma membrane. Clathrin- coated transport vesicles can rapidly and selectively convey nutrient receptors, signaling molecules, ion channels, transporters, and structural proteins off the surface membrane, delivering them to an endosomal sorting station for further processing. The cargo selection process is managed by adaptor proteins, traditionally thought primarily to be the heterotetrameric AP-2 complex. In the previous funding cycle, we provided firm experimental evidence that cargo recognition is expanded by a set of highly-selective clathrin- associated sorting proteins (CLASPs). We also made substantial progress in delineating peptide-based interaction motifs within CLASPs that facilitate direct clathrin and AP-2 binding, and have defined structurally some of the cognate interaction surfaces upon the core coat components. In this application, our goals are to extend this characterization of the molecular principles of clathrin coat operation. On the basis of both Saccharomyces cerevisiae and mammalian live-cell imaging studies, it appears that the time of entry and exit of endocytic factors at the bud site reflects a specific role in the assembly/sorting process. Yet, how this is dictated by the protein-protein interaction elements contained within individual endocytic components is not fully apparent. Using a combination of structural studies, biochemical assays for clathrin assembly, RNA interference and functional uptake assays, ultrastructural analysis, and live-cell microscopy, we will dissect individual protein-protein interactions to unravel their contribution and importance in the coat assembly and sorting process. In particular, we will focus on the clathin-AP-2 beta2 subunit interaction hub, and on Disabled-2 and the autosomal recessive hypercholesterolemia (ARM) protein, two CLASPs involved in low density lipoprotein internalization. Additionally, the concept of compositionally heterogeneous, differentially cargo-selective clathrin coats at the cell surface will be tested experimentally. It is anticipated that fundamental new information regarding the molecular mechanisms that link clathrin coat polymerization to cargo capture at the cell surface will be obtained through these investigations.